Fine grain carriers and medicinal composition prepared with the use of the same

ABSTRACT

The present invention is directed to particulate carriers useful as drug carriers in a drug delivery system (DDS) and to pharmaceutical compositions making use of such carriers. The invention is characterized by the use, as the particulate carrier, of: graft copolymer (A) whose graft chain is poly N-alkylacrylamide chain, poly N-alkylmethacrylamide chain, etc.; and a composition containing a combination of the graft copolymer (A) and at least one graft copolymers selected from the group consisting of graft copolymers (B-1) having polyacrylic acid or polymethacrylic acid as the graft chain and graft copolymers (B-2) having a polyvinyl amine compound as the graft chain.

This application is a Division of application Ser. No. 09/101,804 filedon Aug. 21, 1998, now U.S. Pat. No. 6,100,338, which is a 371,PCT/JP97/00463 filed on Feb. 20, 1997.

TECHNICAL FIELD

The present invention relates to particulate carriers which are usefulas drug carriers in a drug delivery system (DDS) and to pharmaceuticalcompositions containing the carriers.

BACKGROUND ART

In the field of DDS, the term “drug carriers” is used to refer tocarriers that deliver drugs to target organs or cells. When drugcarriers are in the form of particles, they are called particulatecarriers. Particulate carriers are classified into microcapsules,microspheres, nanoparticles, etc. according to their sizes, shapes, andfunctions. Materials for preparing particulate carriers include lipids,polymers, etc.

The terms “microcapsules” and “microspheres” are usually used to referto particles whose diameter is several micrometers. Microcapsules aregenerally considered to embrace a broader category than microspheres.Particles formed of a polymer from the surface to the core are oftendistinguished from microcapsules and are referred to as microspheresthese days.

The term “nanoparticles” has conventionally been used to refer topolymeric colloids prepared through emulsion polymerization, as theparticles size is on the order of several nanometers. However, it hasrecently become common practice to collectively refer to particlescomposed of natural or synthetic polymers, even though prepared throughmethods other than emulsion polymerization, as nanoparticles so long asthe particle diameter is on the order of several nanometers.

Nanoparticles as particulate carriers were first studied for use ascarriers for targeting, for example, anti-cancer agents. In the earlystudies, the primary object of application was injection (L. Grislain etal., International Journal of Pharmaceutics, 15, 335 (1984)). Since themid 1980's, studies of nanoparticles as oral dosage forms have come tobe reported.

When drugs are prepared as the form of nanoparticles and used as oraldosage forms, the following are considered goals to attain: improvementof drugs with poor absorptive characteristic.(P. Maincent et al.,Journal of Pharmaceutical Sciences, 75, 955 (1986); C. Damge et al.,International Journal of Pharmaceutics, 36, 121 (1987)), oral dosageforms of peptide drugs such as insulin (C. Damge et al., Diabetes, 37,246 (1988); P. Couvreur and F. Puisieux, Advanced Drug Delivery Reviews,10, 141 (1993)), oral delivery of vaccines antigen (J. H. Eldrige,Journal of Controlled Release, 11, 205 (1990); P. U. Jani et al.,International Journal of Pharmaceutics, 86, 239 (1992)), and controlledrelease of drugs (B. Hubert et al., Pharmaceutical Research, 8, 734(1991)).

Moreover, like the case of microcapsules, nanoparticles are sometimesused in an attempt to ensure stability of drugs in gastrointestinaltract (M. Rogues et al., Diabetes, 41, 451 (1992)) or to reduceirritation caused by strongly stimulative drugs on gastrointestinalmucosa (N. Ammoury et al., Pharmaceutical Research, 8, 101 (1991)).

Nanoparticles for pharmaceutical use are principally prepared by one ofthe two methods. The first method is a typical microcapsulation method,which is practiced through phase separation or solvent evaporation.

When this method is practiced, there are usually used hydrophobicpolymers that have customarily been used as additives forpharmaceuticals, such as polylactic acid (A. M. Ray et al., Journal ofPharmaceutical Sciences, 83, 845 (1994)), cellulose derivatives (H.Ibrahim et al., International Journal of Pharmaceutics, 87, 239 (1992),or polyacrylate derivatives (E. Allemann et al., International Journalof Pharmaceutics, 87, 247 (1992)).

The other method for the preparation of nanoparticles makes use ofemulsion polymerization (L. Vansnick et al., Pharmaceutical Research, 1,36 (1985); N. Al Khouri Fallouh et al., International Journal ofPharmaceutics, 28, 125 (1986)). In this case, hydrophobic polyvinylcompounds such as polystyrene, polyacrylate, and polymethacrylate areconsidered to serve as the material of the nanoparticles.Polycyanoacrylates are used quite often, especially polyisobutylcyanoacrylate, which is an adhesive for surgical operations.

Drug products are prepared by combining a drug with nanoparticles so asto carry the drug. Drugs to be carried are usually hydrophobiccompounds, because the method for preparing nanoparticles is notsuitable for hydrophilic compounds. Although there have been reportedsome examples in which hydrophilic compounds are transformed intonanoparticles, they are in effect limited to only compounds (e.g.,peptides) that are insoluble in water at a certain pH (YoshiakiKAWASHIMA, The 114th Conference of Japan Pharmaceutical Society, LectureAbstracts Vol. 4, page 9, 1994, Tokyo).

Examples of studies in which the thus-prepared nanoparticle-drugcomplexes are used so as to improve absorption of poor absorptive drugs,to prepare oral dosage forms of peptide drugs, and to control therelease of drugs include the following.

P. Maincent et al. prepared nanoparticles of vincamine, a poorabsorptive hypotensive drug, through use of polyhexyl cyanoacrylate andstudied absorption enhancement effect. However, the absorption rate ofvincamine after transformation into nanoparticles was only 1.6 timesthat before transformation (Journal of Pharmaceutical Sciences, 75, 955(1986)).

C. Damge attempted to prepare oral dosage forms of peptides byencapsulating insulin into nanoparticles through use of polyisobutylcyanoacrylate. However, a slight decrease in blood glucose was observedonly when nanoparticles containing a considerable amount of insulin wereadministered perorally, under fasting, to rats that had experimentallyinduced diabetes (Diabetes, 37, 246 (1988)).

Moreover, B. Hubert et al. studied controlled release of drugs usingdarodipine, a hypotensive drug. However, they were successful only inreducing initial release of the drug by encapsulating the drug intonanoparticles (Pharmaceutical Research, 8, 734 (1991)). There is noreport that controlled release was acheived by nanoparticles.

As described above, there was no particulate carriers that have thesufficient oral absorption enhancement effect of drugs. Accordingly, thepresent invention is directed to a particulate carrier that has anexcellent enhancement effect of drug absorption, and also to apharmaceutical composition containing the carrier.

DISCLOSURE OF THE INVENTION

The present inventors conducted extensive studies, focusing on graftcopolymers as drug carriers particularly the absorption enhancement ofdrugs administered orally. They found that graft copolymers having graftchains composed of polyvinylamine compound show an excellent oralabsorption enhancement effect, and filed a pertinent patent application(Japanese Patent Application Laid-Open (kokai) No. 8-268916). Aftercontinued studies, they unexpectedly found that combinations of one ormore species of graft copolymers having the graft chains composed ofpoly N-alkylacrylamide or poly N-alkylmethacrylamide shown below exhibita remarkably superior oral absorption enhancement effect as compared toconventional graft copolymers, leading to completion of the presentinvention.

Accordingly, the present invention provides a particulate carrierincluding a graft copolymer (A) having structural units of the followingformulae (1) and (2):

wherein

Q¹ is a hydrogen atom, a methyl group, or a cyano group, and

Q² is a hydrogen atom,

wherein

R¹ is a hydrogen atom or a halogenomethyl group,

R² is a C₁-C₁₀ alkyl group,

R³ is a hydrogen atom or a C₁-C₁₀ alkyl group, and

R⁴ is a C₁-C₁₀ alkyl group, provided that the carbon number in total ofR³ and R⁴ is between 3 and 20 inclusive;

wherein

Q³ is a hydrogen atom or a methyl group,

Q⁴ is a group having the following structure:

wherein A¹ is a C₁-C₁₀ alkylene group,

Q⁵ is an oxygen atom or —NH—,

Q⁶ is a C₁-C₁₀ alkylene group,

Q⁷ is an oxygen atom or a sulfur atom,

X¹ is an oxygen atom or two hydrogen atoms,

each of R⁵, R⁷, and R⁸ is a hydrogen atom or a methyl group,

R⁶ is a C₁-C₁₀ alkyl group,

l is a number from 1 to 100, and

each of m and n is a number from 0 to 100.

The present invention also provides a particulate carrier compositioncontaining a composition (graft copolymer composition) of the followingcomponents (a) and (b):

(a) the aforementioned graft copolymer (A); and

(b) one or more graft copolymers selected from the group consisting ofthe following graft copolymers (B-1) and (B-2):

(B-1) a graft copolymer having structural units of the followingformulae (1) and (3):

wherein

Q¹ is a hydrogen atom, a methyl group, or a cyano group, and

Q² is a hydrogen atom,

wherein

R¹ is a hydrogen atom or a halogenomethyl group,

R² is a C₁-C₁₀ alkyl group,

R³ is a hydrogen atom or a C₁-C₁₀ alkyl group, and

R⁴ is a C₁-C₁₀ alkyl group, provided that the carbon number in total ofR³ and R⁴ is between 3 and 20 inclusive;

wherein

Q⁸ is a hydrogen atom or a methyl group,

Q⁹ is a group having the following structure:

wherein A² is a C₁-C₁₀ alkylene group,

Q¹⁰ is an oxygen atom or —NH—,

Q¹¹ is a C₁-C₁₀ alkylene group,

Q¹² is an oxygen atom or a sulfur atom,

X² is an oxygen atom or two hydrogen atoms,

each of R⁹, and R¹⁰ is a hydrogen atom or a methyl group,

R¹¹ is a C₁-C₁₀ alkyl group, and

p and q are independently numbers from 0 to 100 such that the sum p+q isequal to or more than 1;

(B-2) a graft copolymer having structural units of the followingformulae (1) and (4):

wherein

Q¹ is a hydrogen atom, a methyl group, or a cyano group, and

Q² is a hydrogen atom,

wherein

R¹ is a hydrogen atom or a halogenomethyl group,

R² is a C₁-C₁₀ alkyl group,

R³ is a hydrogen atom or a C₁-C₁₀ alkyl group, and

R⁴ is a C₁-C₁₀ alkyl group, provided that the carbon number in total ofR³ and R⁴ is between 3 and 20 inclusive;

wherein

Q¹³ is a hydrogen atom or a methyl group,

Q¹⁴ is a group having the following structure:

 wherein A³ is a C₁-C₁₀ alkylene group,

Q¹⁵ is an oxygen atom or —NH—,

Q¹⁶ is a C₁-C₁₀ alkylene group,

Q¹⁷ is an oxygen atom or a sulfur atom,

X³ is an oxygen atom or two hydrogen atoms,

each of R¹² and R¹³ is a hydrogen atom or a methyl group,

R¹⁴ is a C₂-C₁₁alkanoyl group, and s and t are independently numbersfrom 0 to 100 such that the sum s+t is equal to or more than 1.

The present invention also provides a pharmaceutical compositioncontaining a drug and the aforementioned graft copolymer (A), or a drugand the aforementioned graft copolymer composition containing thecomponents (a) and (b).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a proton NMR chart of the N-isopropylacrylamide macromonomersynthesized in Reference Example 1-2.

FIG. 2 is a proton NMR chart of the graft copolymer synthesized inReference Example 1-3, in which the hydrophobic backbone was consistedof polystyrene and the hydrophilic branches were consisted of polyN-isopropylacrylamide.

FIG. 3 is a proton NMR chart of the macromonomer that was synthesized inReference Example 2-2, which was consisted of random-copolymer ofN-isopropylacrylamide and acrylamide.

FIG. 4 is a proton NMR chart of the macromonomer that was synthesized inReference Example 4-2, which was consisted of random-copolymer ofN-isopropylacrylamide and acrylamide.

FIG. 5 is a proton NMR chart of the macromonomer that was synthesized inReference Example 6-2, which was consisted of random-copolymer ofN-isopropylacrylamide and acrylamide.

FIG. 6 is a graph showing the plasma phenolsulfonphthalein concentrationas a function of time (mean±S.D.), after phenolsulfophthalein wasduodenally administered to rats in Example 2.

FIG. 7 is a graph showing the change of ionized calcium concentration inblood (mean±S.E.) as a function of time, after salmon calcitonin wasperorally administered to rats in Example 4.

FIG. 8 is a graph showing the change of analgetic effect (mean±S.E.)with the passage of time when opioid peptide was perorally administeredto mice in Example 6.

FIG. 9 is a graph showing the change of ionized calcium concentration inblood (mean±S.E.) as a function of time, after salmon calcitonin wasperorally administered to rats in Example 8.

FIG. 10 is a graph showing the change of ionized calcium concentrationin blood (mean±S.E.) as a function of time, after salmon calcitonin wasperorally administered to rats in Example 10.

FIG. 11 is a graph showing the change of ionized calcium concentrationin blood (mean±S.E.) as a function of time, after salmon calcitonin wasperorally administered in divided portions to rats in Example 11.

BEST MODE FOR CARRYING OUT THE INVENTION

The graft copolymers which may be used in the particulate carriers andin the pharmaceutical compositions of the present invention arecategorized into the following three groups; namely, graft copolymers(A) having structural units (1) and (2), graft copolymers (B-1) havingstructural units (1) and (3), and graft copolymers (B-2) havingstructural units (1) and (4).

The proportion of graft chains present in these graft copolymers is notparticularly limited. However, from the viewpoint of the enhancementeffect of drug absorption, the mole fraction of the structural unit offormula (2), (3), or (4) should be between 0.001 and 1.

Next will be described the symbols used in formulae (1), (2), (3), and(4), which represent structural units composing of the graft copolymersof the present invention.

In formula (1), examples of the halogenomethyl groups represented by R¹include a chloromethyl group, bromomethyl group, an iodomethyl groupetc. The C₁-C₁₀ alkyl groups represented by R², R³, and R⁴ may be linearor branched; specific examples of which include a methyl group, ethylgroup, n-propyl group, isopropyl group, n-butyl group, t-butyl group,n-pentyl group, n-hexyl group etc. Of these, R² is preferably C₁-C₅alkyl, with a methyl group, ethyl group, and an isopropyl group beingparticularly preferred. There are two cases: one in which R³ is ahydrogen atom and R⁴ is an alkyl group, and one in which R³ and R⁴ areboth alkyl groups. In either case, the carbon number in total is between3 and 20 inclusive. For example, when R³ is a hydrogen atom, R⁴ is analkyl group having 3-10 carbon atoms, and when R³ and R⁴ are both alkylgroups, R³ and R⁴ are alkyl groups such that the carbon number in totalis between 3 and 20 inclusive.

Among the structural units represented by formula (1), the structuralunit of the following formula (1a):

(wherein Q¹ and R¹ have the same meanings as defined above) ispreferred, and the structural unit of the following formula (1b):

(wherein R¹ has the same meaning as defined above) is particularlypreferred.

In formulae (2), (3), and (4), the C₁-C₁₀ alkylene groups represented byA¹, A², A³, Q⁶, Q¹¹, and Q¹⁶ may be linear or branched; specificexamples of which include a methylene group, ethylene group,trimethylene group, hexamethylene group, propylene group,(ethyl)ethylene group, (dimethyl)ethylene group etc. Of these, C₁-C₅linear or branched alkylene groups are preferred.

The C₁-C₁₀ alkyl groups represented by R⁶ and R¹¹ may be linear orbranched, specific examples of which include a methyl group, ethylgroup, n-propyl group, isopropyl group, n-butyl group, t-butyl group,n-pentyl group, an n-hexyl group etc. Of these, R⁶ is preferably C₃-C₁₀branched alkyl groups, in which isopropyl groups are particularlypreferred. R¹¹is preferably C₁-C₈ linear or branched alkyl groups, inwhich a methyl group, ethyl group, isopropyl group, t-butyl group, andan n-hexyl group are particularly preferred.

The C₂-C₁₁ alkanoyl groups represented by R¹⁴ may be linear or branched.Preferably, they have 2 to 6 carbon atoms. An acetyl group, propionylgroup, and a butyryl group are particularly preferred.

In formula (2), because each of m and n may be zero, there are cases inwhich the structural unit of formula (2) takes the structure representedby one of the following formulae (2a), (2b), (2c) and (2d):

wherein Q³, Q⁴, Q⁵, Q⁶, Q⁷, X¹, R⁵, R⁶, R⁷, R⁸, and 1 have the samemeanings as defined above.

Of the structural units of formula (2), preferred are those of formula(2e):

(wherein Q⁵, Q⁶, Q⁷, X¹, R⁵, R⁶, R⁷, R⁸, l, m, and n have the samemeanings as defined above), and particularly preferred are those offormulae (2f) and (2g):

(wherein R⁶, l, m, and n have the same meanings as defined above).

In formula (3), because each of p and q may be zero, there are cases inwhich the structural unit of formula (3) takes the structure representedby one of the following formulae (3a), (3b), and (3c):

wherein Q⁸, Q⁹, Q¹⁰, Q¹¹, Q¹², X², R⁹, R¹⁰ and R¹¹ have the samemeanings as defined above.

Of the structural units of formula (3), preferred are those of formula(3d):

(wherein Q¹⁰, Q¹¹, Q¹², X², R⁹, R¹⁰, R¹¹, p, and q have the samemeanings as defined above), and particularly preferred are those offormulae (3e) and (3f):

(wherein R¹⁰, p, and q have the same meanings as defined above).

In formula (4), because each of s and t may be zero, there are cases inwhich the structural unit of formula (4) takes the structure representedby one of the following formulae (4a), (4b), (4c):

wherein Q¹³, Q¹⁴, Q¹⁵, Q¹⁶, Q¹⁷, X³, R¹², R¹³, and R¹⁴ have the samemeanings as defined above.

Of the structural units of formula (4), preferred are those of formula(4d):

(wherein Q¹⁵, Q¹⁶, Q¹⁷, X³, R¹², R¹³, R¹⁴, s, and t have the samemeanings as defined above), and particularly preferred are those offormulae (4e) and (4f):

(wherein R¹⁴, s, and t have the same meanings as defined above).

In the above-described graft copolymers, the recurring unit in eachgraft chain (such as N-alkylacrylamide, N-alkylmethacrylamide,acrylamide, methacrylamide, acrylic acid, vinylamine,N-alkanoylvinylamine etc.) may be of the random type or the block type.Also, bonding between the structural unit of formula (1) and thestructural unit of any one of formula (2), (3), or (4) may be of therandom type or the block type.

Of the aforementioned graft copolymers of formula (A), those fallingwithin the following three categories are novel: graft copolymerscomposing of structural units of formula (1) and formula (2b); graftcopolymers composing of structural units of formula (1) and formula(2c); and graft copolymers composing of structural units of formula (1)and formula (2d).

The graft copolymers may be prepared by, for example, synthesizing amacromonomer corresponding to the structural unit of formula (2), (3),or (4), and subsequently copolymerizing the resultant macromonomer and avinyl compound that corresponds to formula (1).

The method for preparing the graft copolymers will next be described indetail.

A macromonomer which corresponds to the structural unit of formula (2),(3), or (4) may be readily prepared through radical polymerization, inthe presence of a chain transfer agent having an amino group, a hydroxylgroup, or a carboxyl group in the molecule, of one or more monomers (forexample, an alkylacrylamide derivative, and an alkylmethacrylamidederivative) corresponding to the recurring unit of any one of theformulae (2) through (4), to thereby synthesize one or more polymers orcopolymers each having an amino, hydroxyl, or carboxyl group (forexample, polymers or copolymers of an alkylacrylamide derivative or analkylmethacrylamide derivative) in the terminal, and subsequentlyreacting the resultant polymers or copolymers with a vinyl monomer suchas vinyl benzyl halide or alkyl methacrylate dioxide.

Polymerization of one or more monomers such as an alkylacrylamidederivative or an alkylmethacrylamide derivative is performed in thepresence of a chain transfer agent and a radical polymerizationinitiator. During polymerization, solvents may or may not be present.Presence of a solvent is preferred from the viewpoint of controlledreaction and convenience in operation. Examples of solvents include, butare not limited to, water, alcohols, dimethylformamide, and benzene.Examples of chain transfer agents include mercaptoalkylamines,mercaptoalkanols, omega-mercaptocarboxylic acids, alkylene glycols etc.Of these, 2-mercaptoethylamine, 2-mercaptoethanol, andbeta-mercaptopropionic acid are preferred. Radical polymerizationinitiators include azobisisobutyronitrile, benzoyl peroxide, andammonium persulfate. Of these, azobisisobutyronitrile and benzoylperoxide are preferred.

The reaction of a vinyl monomer and one or more polymers or copolymerseach having an amino, hydroxyl, or carboxyl group (for example, polymersor copolymers of an alkylacrylamide derivative or an alkylmethacrylamidederivative) in the terminal may be easily performed through aconventional acid amide reaction, etherification, or esterification.Preferred vinyl monomers include chloromethylstyrene and propylenemethacrylate dioxide.

For example, a random copolymer composed of an alkylacrylamidederivative or an alkylmethacrylamide derivative having a hydroxyl groupin the terminal and acrylamide or methacrylamide may be reacted at atemperature between 0 and 100° C. with chlorostyrene in a solvent suchas dimethylformamide in the presence of an aqueous 50% KOH aqueoussolution and, if necessary, a phase transfer catalyst.

When the thus-prepared macromonomer which corresponds to the structuralunit of formula (2), (3), or (4) is polymerized, or copolymerized withvinyl compounds corresponding to formula (1) which are able toradical-polymerize, the aforementioned graft copolymer is obtained.

Examples of the vinyl compounds include styrene, halomethylstyrene,methyl acrylate, methyl methacrylate, isobutyl cyanoacrylate,acrylonitrile, acrylamide, and vinyl acetate. Of which styrene,halomethylstyrene, methyl acrylate, and methyl methacrylate arepreferred.

Of the above-described graft copolymers, those of formula (2b), (2c),(2d), (3a), (3b), (4a), or (4b), which have an acid amide group, acarboxyl group, or a primary amino group in the graft chain, may also beprepared by polymerizing a macromonomer having a structural unit offormula (2a), (3c), and/or (4c), or copolymerizing each of them with aaforementioned vinyl compounds, and subsequently hydrolyzing theresultant product to a suitable extent by a known method.

By varying the degree of polymerization, it is possible to obtainamphipathic graft copolymers which are soluble in water, alcohol,chloroform, dimethylsulfoxide, etc.

Particles composed of these graft copolymers are obtained throughdispersion polymerization of a hydrophobic monomer and a hydrophilicmacromonomer which corresponds to the structural unit of formula (2),(3), or (4) and through subsequent hydrolysis, which may be performed ifnecessary. Each of the resultant particles has a form in which thehydrophilic macromonomer is localized in the outer of the particles andthe inner of the particles is constituted by the hydrophobic polymer.

Since the surface of the thus-prepared particles are hydrophilic,hydrophilic drugs can be effectively incorporated in the particles. Onthe other hand, there is the hydrophobic interaction between thehydrophobic drugs and the inner layer composed of a hydrophobic polymer.Hydrophobic drugs may also incorporated to the outer of particles,making use of the amphipathic property of the outer. In other words,since the particles of the present invention are considered to haveability to incorporate drugs effectively without particular dependenceon properties of the drugs, the particles of the invention are useful asparticulate carriers.

In the present invention, a preferred carrier for drugs is a compositioncontaining a mixture of a graft copolymer (A) (may be referred to ascomponent (a)) and one or more members selected from the groupconsisting of graft copolymers (B-1) and graft copolymers (B-2) (may bereferred to as component (b)). (Hereafter, the composition composed of acomponent (A) and a component (B) may be referred to as a graftcopolymer composition.) Because this graft copolymer composition has twoor more kinds of graft chains derived from a hydrophilic macromonomer,it is considered that drugs are effectively incorporated withnanoparticles and are protected against the enzyme in thegastrointestinal tract, and that absorption of drugs by the intestinaltract is enhanced.

The ratio of amounts of components (a) to (b) contained in the drugcarrier of the invention is preferably from 1,000:1 to 1:1,000, andparticularly preferably from 100:1 to 1:100.

In order to use the above-described graft copolymers as particulatecarriers, the graft copolymers are prepared to have the form ofmicrocapsules, microspheres, or nanoparticles.

Microcapsules and microspheres may be obtained by use of conventionalmethods. Nanoparticles can be obtained by use of a macromonomer methoddeveloped by Akashi et al. (Die Angewandte, Macromolekulare Chemie, 132,81 (1985); Polymer Journal, 24, 959 (1992); Chemical Engineering, page505, 1994) and dispersion polymerization, to thereby obtainnanoparticles in which a hydrophilic macromonomer is localized in theouter of each particle and the inner of each particle is formed of ahydrophobic polymer.

The particle diameter of nanoparticles varies in accordance with themolecular weight of the macromonomer, reaction conditions under whichthe macromonomer is prepared, and other factors. When suitable settingsand conditions are selected, it is possible to obtain microspheres whosediameter is on the order of micrometers.

As described above, since the graft copolymers or graft copolymercompositions are useful as drug carriers, when the graft copolymers orgraft copolymer compositions are mixed with drugs, pharmaceuticalcompositions exhibiting excellent peroral absorption enhancement effectcan be obtained. It is considered that in a drug composition prepared bymixing a graft copolymer or graft copolymer composition with a drug, thegraft copolymer or graft copolymer composition and the drug togetherform a complex. The driving power for the formation of a complex isconsidered to be electrostatic interaction, hydrogen bonding (i.e.,interaction with hydrophilic functional groups on the outer ofnanoparticle), hydrophobic interaction (attraction to the inner of theparticle).

Drugs which are useful in the preparation of the pharmaceuticalcomposition of the present invention are not particularly limited, andmay be hydrophilic or hydrophobic. There are the drugs that are expectedto be controlled the release or to be enhanced the absorption.

Drugs that are expected to be controlled the release include 1) drugshaving a short half-life in blood and 2) drugs having a narrow optimumtherapeutic range. Drugs that are expected to be enhanced theabsorption, or in other words poor absorptive drugs, include 3) drugshaving a low membrane permeability due to their high water-solubility,4) drugs whose efficacy is hindered from being exerted due todegradation in the gastrointesinal tracts, low absorptivecharacteristics via the gastrointesinal tracts, etc. and 5) vaccines.

1) Examples of drugs having a short half-life in blood includeisosorbide, papaverine, nitroglycerin, ketoprofen, diltiazem,propranolol, isoproterenol, isotipenzyl, aspirin, pindrol, nifedipine,acetazolamide, cephalexin, cefaclor, quinidine, and procain amide.

2) Examples of drugs having a narrow optimum therapeutic range includepilocarpine, theophylline, scopolamine, methyl scopolamine,chlorpheniramine, phenylephrine, trihexyphenidyl, carbetapentane,perphenazine, noscapine, thioridazine, dimethindene, pyridostigmine, andtriprolidine.

3) Examples of drugs having a low membrane permeability due to theirhigh water-solubility include phenolsulfonphthalein, salicylic acid andits derivatives, barbituric acid and its derivatives, quaternaryammonium salts such as tubocurarine and suxamethonium, sulfa agents suchas sulfanyl acid, sulfanyl acetamide, and sulfaguanidine, quinine,ephedrin, tolazoline, procainamide, atenolol, and chlorothiazide.

4) Examples of drugs whose efficacy is hindered from being exerted dueto degradation in the gastrointestinal tracts, low absorptivecharacteristics via the gastrointestinal tracts include peptides; morespecifically, interferon, interleukin, erythropoietin, insulin,neocarcinostatin, parathormone, opioid peptides and calcitonin.

5) Examples of vaccines include those which are considered useful whenadministered by the oral route. Specific examples of the vaccinesinclude influenza HA vaccine, hepatitis B vaccine, and polio vaccine.

Examples of antigens for the preparation of vaccines include a varietyof proteins from: viruses such as influenza A virus, influenza B virus,influenza C virus, rotavirus, cytomegalovirus, RS virus, adenovirus,AIDS virus (HIV), hepatatis A virus, hepatitis B virus, hepatitis Cvirus, varicella-zoster virus, herpes simplex virus (type 1 and type 2),adult T cell leukemia virus (ATLV), coxsackie virus, enterovirus,exanthema subtium virus, measles virus, rubella virus, mumps virus,polio virus, Japanese encephalitis virus, and rabies virus; bacteriasuch as Streptococcus caries, Vibrio cholerae, Haemophilus influenzae,Streptococcus pneumoniae, Bordetella pertussis, Corynebacteriumdiphtheriae, and Clostridium tetani; rickettsias such as chlamydia; andprotozoas such as malaria plasmodium. Moreover, the above-listedviruses, bacteria, rickettsias, and protozoas by themselves may be usedas antigens after their phathogenity has been weakened. Among theabove-listed drugs, those that are expected to be enhanced absorption(i.e., poor absorptive drugs) are preferably used in the presentinvention. Peptides are more preferred, and opioid peptide andcalcitonin are particularly preferred.

It is considered that, when the above-described complexes areadministered perorally, most drugs are delivered to the vicinity of themicrovilli of the gastrointestinal tract while retaining their complexforms, since the particles of the complexes are very small (J. Kreuteret al., International Journal of Pharmaceutics, 55, 39 (1989)).Moreover, since the graft copolymers used in the present invention havehydrophilic groups on the outer of them, the compatibility of thecomplexes with the membranes of the gastrointestinal tract is high.

In short, it is considered that a particulate carrier composed of thegraft copolymer or graft copolymer composition of the present invention,particularly a complex of nanoparticle and drug, can accumulate the drugat a high concentration in the vicinity of the membranes. As a result,absorption of poor absorptive drugs can be improved.

Moreover, peptides and similar drugs which are easily degraded bydigestive enzymes in the gastrointestinal tract can be protected fromattacks by the enzymes when formulated to have the form of particles.

In addition, the complexes of particles and drugs, having thecompatibility with the membranes, are considered to decrease the transittime of drugs in the gastrointestinal tract. Consequently, controlledrelease of the drugs is expected, as the drugs retain the absorptionsite, i.e., in the gastrointestinal tract, over prolonged periods.

In pharmaceutical compositions of the present invention, the ratio ofthe amount of the graft copolymer or graft copolymer composition to theamount of the drug is adjusted in accordance with the drug to beemployed.

In practice, a mixture or a complex of the graft copolymer or graftcopolymer composition and a drug are formulated into a drug preparationby known method and administered perorally. Alternatively, the mixtureor the complex of the graft copolymer or graft copolymer composition anda drug may be encapsulated in soft capsules.

The physical form of the drug preparation is not particularly limited.The drugs preparation may take the solid formulation such as tablets,granules, powders, and capsules etc; or the liquid formulation such assyrups, elixirs, suspensions, emulsions etc. When these drugpreparations are manufactured, it goes without saying that customaryadditives such as excipients, binders, lubricants, and disintegrants mayalso be mixed.

The drug absorption enhancement effect of the pharmaceuticalcompositions of the present invention is not reduced even underconditions of low pH such as at a pH of 1.2. Moreover, this effect isnot reduced at the body temperature of 40° C. Therefore, thepharmaceutical composition of the present invention are particularlyuseful for peroral administrations.

As will be described in the Example section hereinbelow, the drugabsorption enhancement effect of the pharmaceutical compositions of thepresent invention is greatly improved when the compositions areadministered in a divided manner with intervals of a certain period.Accordingly, by suitably designing the manner of dosage or means offormulation of the drug preparations (for example, by administering asingle dose in two or more divided portions, or by incorporating a rapidrelease formulation and a slow release formulation in combination withina single drug preparation), the drug absorption enhancement effect ofthe pharmaceutical compositions of the present invention is even furtherimproved.

EXAMPLES

The present invention will next be described by way of referenceexamples, working examples, and test examples, which should not beconstrued as limiting the invention.

Reference Example 1 Graft copolymer having Poly N-isopropylacrylamide asthe Graft Chain (Graft Copolymer (A-1))

1-1. Synthesis of an N-isopropylacrylamide oligomer

N-isopropylacrylamide (20 g, Kojin), 2,2′-azobisisobutyronitrile (AIBN)(0.6 g), and 2-mercaptoethanol (0.20 g) were dissolved in ethanol (80ml). The solution was polymerized for 7 hours at 60° C. in an atmosphereof nitrogen. When polymerization was completed, the solvent contained ina reaction mixture was removed by use of an evaporator. The prepolymerwas re-dissolved in distilled water, heated, and centrifugally separatedat a temperature of 60° C. or higher for purification, and the resultantpurified product was freeze-dried. The yield of the polymer was 83%. Thenumber average molecular weight (Mn) of the polymer determined by gelpermeation chromatography (GPC) was 3,400.

1-2. Synthesis of an N-isopropylacrylamide Macromonomer

The N-isopropylacrylamide oligomer (8 g) obtained in Step 1-1 wasdissolved in dimethylformamide (DMF) (50 ml). To the solution was added50% KOH (3.3 g), and the resultant mixture was stirred for 30 minutes at30° C. Tetrabutylphosphate bromide (500 mg) was added, and subsequently,chloromethylstyrene (4.65 g) was added. The mixture was allowed to reactfor 72 hours at 30° C. The precipitation were removed by filtration, andthe reaction mixture was dialyzed and freeze-dried. The yield of themacromonomer was 82%. The number average molecular weight (Mn) of themacromonomer determined by GPC was 3,500. The macromonomer was solublein water and in ethanol.

FIG. 1 is the proton NMR chart obtained for the thus-obtainedN-isopropylacrylamide macromonomer in DMSO-D⁶.

The chart shows peaks attributed to the vinyl protons of styrene(δ=5.3-6.0 ppm and 6.6-7.0 ppm) and a peak attributed to the proton ofthe benzene nucleus in the vicinity of 7.5 ppm.

1-3. Copolymerization of an N-isopropylacrylamide Macromonomer andStyrene (Synthesis of a Graft Copolymer (A-1)); and Preparation ofNanoparticles)

The macromonomer obtained in Step 1-2 (Mn=3,500) (635 mg), styrene (520mg), and AIBN (8.5 mg) were dissolved in ethanol (5 ml). The solutionwas allowed to react for 24 hours at 60° C. After polymerization, theunreacted substance and the solvent were removed by dialysis, and thepolymer was freeze-dried. The polymer was soluble in chloroform and inDMSO. The particle diameter of the graft copolymer was 430 nm, asmeasured by the dynamic light scattering spectrophotometry.

FIG. 2 is the proton NMR chart obtained for the graft copolymer inDMSO-D⁶.

In the chart, the vinyl protons of styrene (δ=5.3-6.0 ppm and 6.6-7.0ppm) observed in the aforementioned macromonomer disappeared, and intheir place were observed peaks attributed to the protons of themethylene group of styrene (δ=1.8-2.6 ppm) and a peak attributed to theproton of the benzene nucleus in the vicinity of 7.5 ppm.

Reference Example 2 Graft Copolymer which has a Random CopolymerComposed of N-isopropylacrylamide and Acrylamide as the Graft Chain (theProportion of N-isopropylacrylamide Contained in the Graft Chain Being53% ) (Graft Copolymer (A-2))

2-1. Synthesis of an Oligomer of a Random Copolymer Composed ofN-isopropylacrylamide and Acrylamide

An N-isopropylacrylamide monomer (8.70 g, 75 mmol) and an acrylamidemonomer (1.44 g, 25 mmol) were dissolved in ethanol (50 ml).2-Mercaptoethanol (0.273 g, 3.50 mmol) as a chain transfer agent andazobisisobutyronitrile (0.164 g, 1.00 mmol) as a radical polymerizationinitiator were added. Polymerization was performed for 6 hours at 60° C.in an atmosphere of nitrogen, to thereby synthesize the title oligomer.After reaction, the solvent was evaporated and the residue, after beingdissolved in acetone, was allowed to precipitate in hexane to therebyrecover the product. The reprecipitation was conducted several times soas to purify the oligomer. The molecular weight (Mn) of the oligomer was3,100, as determined by GPC.

2-2. Synthesis of a Macromonomer of a Random Copolymer Composed ofN-isopropylacrylamide and Acrylamide

The oligomer (4 g, 1.29 mmol) obtained in 2-1 was dissolved indimethylformamide (50 ml). To the solution were added sodium hydride(0.062 g, 2.58 mmol) and tetrabutylphosphonium bromide (2.189 g, 6.45mmol) as a phase transfer catalyst, and the mixture was stirred for 60minutes. Thereafter, p-chloromethylstyrene (4.10 g, 26.7 mmol) wasadded, and the mixture was stirred for 48 hours at 30° C. After beingstirred, the solvent was evaporated and the residue, after beingdissolved in acetone, was allowed to precipitate in hexane to therebyrecover the product. The reprecipitation was conducted several times forpurification. The introduction rate of vinyl benzyl group was calculatedbased on the ¹H-NMR data. As a result, it was confirmed that vinylbenzyl group had been almost quantitatively introduced (FIG. 3). Themolecular weight (Mn) of the obtained macromonomer was determined to be4,600 by GPC.

2-3. Copolymerization of Styrene and a Macromonomer of a RandomCopolymer Composed of N-isopropylacrylamide and Acrylamide (Synthesis ofa Graft Copolymer (A-2); and Preparation of Nanoparticles)

The macromonomer obtained in Step 2-2 (0.300 g, 0.065 mmol) and styrene(0.500 g, 4.80 mmol) were dissolved in ethanol (5 ml).Azobisisobutyronitrile (0.008 g, 0.049 mmol) as a radical polymerizationinitiator was added, and polymerization was performed in a deaeratedsealed tube for 48 hours at 60° C. After reaction, the contents of thetube were subjected to several repetitions of centrifugal separation andredispersal in ethanol. In the last step, the graft copolymer wasdispersed in water to thereby conclude purification. Measurement by thedynamic light scattering spectrophotometry revealed that the averageparticle size was 494 nm.

Reference Example 3 Graft Copolymer which has a Random CopolymerComposed of N-isopropylacrylamide and Acrylic Acid as the Graft Chain(the Proportion of N-isopropylacrylamide Contained in the Graft ChainBeing 53%) (Graft Copolymer (A-3))

The nanoparticles obtained in Step 2-3 were dispersed in 2N-HCl, andthen hydrolyzed for 12 hours at 95° C., to thereby substitute theacrylamide group of the macromonomer into acrylic acid group. Afterreaction, dialysis was performed for purification. The acrylamidepresent in the outer of the nanoparticles was confirmed by IR to havebeen hydrolyzed. Thus, the title graft copolymer was obtained. Theaverage particle size was found to be 311 nm by the dynamic lightscattering spectrophotometry.

Reference Example 4 Graft Copolymer which has a Random CopolymerComposed of N-isopropylacrylamide and Acrylamide as the Graft Chain (theProportion of N-isopropylacrylamide Contained in the Graft Chain Being25%) (Graft Copolymer (A-4))

4-1. Synthesis of an Oligomer of a Random Copolymer Composed ofN-isopropylacrylamide and Acrylamide

An N-isopropylacrylamide monomer (3.48 g, 30 mmol) and an acrylamidemonomer (4.03 g, 70 mmol) were dissolved in a solvent mixture (50 ml) ofethanol and water (1:1 (v/v)). 2-Mercaptoethanol (0.273 g, 3.50 mmol) asa chain transfer agent and azobisisobutyronitrile (0.164 g, 1.00 mmol)as a radical polymerization initiator were added. Polymerization wasperformed for 6 hours at 60° C. in an atmosphere of nitrogen, to therebysynthesize the title oligomer. After reaction, the solvent wasevaporated and the residue, after being dissolved in acetone, wasallowed to precipitate in hexane to thereby recover the product. Thereprecipitation was conducted several times so as to purify theoligomer. The molecular weight (Mn) of the obtained oligomer wasdetermined to be 2,100 by GPC.

4-2. Synthesis of a Macromonomer of a Random Copolymer Composed ofN-isopropylacrylamide and Acrylamide

The oligomer (3.5 g, 1.66 mmol) obtained in Step 4-1 was dissolved indimethylformamide (50 ml). To the solution were added sodium hydride(0.080 g, 3.32 mmol) and tetrabutylphosphonium bromide (2.82 g, 8.30mmol) as a phase transfer catalyst, and the mixture was stirred for 60minutes. Thereafter, p-chloromethylstyrene (3.58 g, 23.3 mmol) wasadded, and the mixture was stirred for 48 hours at 30° C. Afterreaction, the solvent was evaporated and the residue, after beingdissolved in acetone, was allowed to precipitate in hexane to therebyrecover the product. The reprecipitation was conducted several times forpurification. The introduction rate of vinyl benzyl group was calculatedbased on the ¹H-NMR data. As a result, it was confirmed that vinylbenzyl group had been introduced almost quantitatively (FIG. 4). Themolecular weight (Mn) of the obtained macromonomer was determined to be2,100 by GPC.

4-3. Copolymerization of Styrene and a Macromonomer of a RandomCopolymer Composed of N-isopropylacrylamide and Acrylamide (Synthesis ofa Graft Copolymer (A-4); and Preparation of Nanoparticles)

The macromonomer obtained in Step 4-2 (0.300 g, 0.065 mmol) and styrene(0.550 mg, 5.28 mmol) were dissolved in a solvent mixture (5 ml) ofethanol and water (1:1 (v/v)). Azobisisobutyronitrile (0.0089 g, 0.0542mmol) as a radical polymerization initiator was added, andcopolymerization was performed in a deaerated sealed tube for 48 hoursat 60° C. After reaction, the contents of the tube were subjected toseveral repetitions of centrifugal separation and redispersal inethanol. In the last step, the graft copolymer product was dispersed inwater to thereby conclude purification. Measurement by the dynamic lightscattering spectrophotometry revealed that the average particle size was347 nm.

Reference Example 5 Graft Copolymer which has a Random CopolymerComposed of N-isopropylacrylamide and Acrylic Acid as the Graft Chain(the Proportion of N-isopropylacrylamide Contained in the Graft ChainBeing 25%) (Graft copolymer (A-5))

The nanoparticles obtained in Step 4-3 were dispersed in 2N-HCl, andthen hydrolyzed for 12 hours at 95° C., to thereby substitute theacrylamide group of the macromonomer into acrylic acid group. Afterreaction, dialysis was performed for purification. The acrylamidepresent in the outer of the nanoparticles was confirmed by IR to havebeen hydrolyzed. Thus, the title graft copolymer was obtained. Theaverage particle size determined by the dynamic light scatteringspectrophotometry was 482 nm.

Reference Example 6 Graft copolymer which has a random copolymercomposed of N-isopropylacrylamide and acrylamide as the graft chain (theproportion of N-isopropylacrylamide contained in the graft chain being68%) (Graft copolymer (A-6))

6-1. Synthesis of an Oligomer of a Random Copolymer Composed ofN-isopropylacrylamide and Acrylamide

An N-isopropylacrylamide monomer (12.75 g, 112.7 mmol) and an acrylamidemonomer (1.82 g, 16.1 mmol) were dissolved in ethanol (50 ml).2-Mercaptoethanol (0.351 g, 4.50 mmol) as a chain transfer agent andazobisisobutyronitrile (0.211 g, 1.29 mmol) as a radical polymerizationinitiator were added. Polymerization was performed for 6 hours at 60° C.in an atmosphere of nitrogen, to thereby synthesize the title oligomer.After reaction, the solvent was evaporated and the residue, after beingdissolved in acetone, was allowed to precipitate in hexane to therebyrecover the product. The reprecipitation was conducted several times soas to purify the oligomer. The molecular weight (Mn) of the obtainedoligomer was 4,400, as determined by GPC.

6-2. Synthesis of a Macromonomer of a Random Copolymer Composed ofN-isopropylacrylamide and Acrylamide

The oligomer (3 g, 0.66 mmol) obtained in Step 6-1 was dissolved indimethylformamide (50 ml). To the solution were added sodium hydride(0.032 g, 1.32 mmol) and tetrabutylphosphonium bromide (1.12 g, 3.30mmol) as a phase transfer catalyst, and the mixture was stirred for 60minutes. Thereafter, p-chloromethylstyrene (2.97 g, 19.3 mmol) wasadded, and the mixture was stirred for 48 hours at 30° C. Afterreaction, the solvent was evaporated and the residue, after beingdissolved in acetone, was allowed to precipitate in hexane to therebyrecover the product. The reprecipitation was conducted several times forpurification. The introduction rate of vinyl benzyl group was calculatedbased on the ¹H-NMR data. As a result, it was confirmed that vinylbenzyl group had been introduced almost quantitatively (FIG. 5). Themolecular weight (Mn) of the obtained macromonomer was determined to be7,200 by GPC.

6-3. Copolymerization of Styrene and a Macromonomer of a RandomCopolymer Composed of N-isopropylacrylamide and Acrylamide (GraftCopolymer (A-6); and Preparation of Nanoparticles).

The macromonomer obtained in Step 6-2 (0.374 g, 0.052 mmol) and styrene(0.650 mg, 6.25 mmol) were dissolved in ethanol (5 ml).Azobisisobutyronitrile (0.010 g, 0.063 mmol) as a radical polymerizationinitiator was added, and copolymerization was performed in a deaeratedsealed tube for 48 hours at 60° C. After reaction, the contents of thetube were subjected to several repetitions of centrifugal separation andredispersal in ethanol. In the last step, the graft copolymer productwas dispersed in water to thereby conclude purification. Measurement bythe dynamic light scattering spectrophotometry revealed that the averageparticle size was 253 nm.

Reference Example 7 Graft Copolymer which has a Random CopolymerComposed of N-isopropylacrylamide and Acrylic Acid as the Graft Chain(the Proportion of N-isopropylacrylamide Contained in the Graft ChainBeing 68%) (Graft Copolymer (A-7))

The nanoparticles obtained in Step 6-3 were dispersed in 2N-HCl, andthen hydrolyzed for 12 hours at 95° C., to thereby substitute theacrylamide group of the macromonomer chain into acrylic acid group.After reaction, dialysis was performed for purification. The acrylamidepresent in the outer of the nanoparticles was confirmed by IR to havebeen hydrolyzed. Thus, the title graft copolymer was obtained. Theaverage particle size determined by the dynamic light scatteringspectrophotometry was 769 nm.

The structure of the graft copolymers synthesized in Reference Examples3, 5, and 7 is shown below.

Reference Example 8 Preparation of a Graft Copolymer havingpoly-tert-butylmethacrylate as the Graft Chain (Graft Copolymer (B-1-1))

8-1. Synthesis of a tert-butylmethacrylate (t-BMA) Oligomer

A tert-butylmethacrylate monomer (25.02 g, 175.8 mmol) was dissolved intetrahydrofuran (THF) (50 ml). 2-Mercaptoethanol (0.345 g, 4.42 mmol) asa chain transfer agent and azobisisobutyronitrile (AIBN) (0.288 g, 1.76mmol) as a radical polymerization initiator were added. Polymerizationwas performed for 6 hours at 60° C. in an atmosphere of nitrogen, tothereby synthesize a t-BMA oligomer. After reaction, the reactionsubstance was purified by a series of reprecipitation through use of amixture of methanol and water (1:1). The molecular weight (Mn) of theobtained polymer was determined to be 3,620 by GPC.

8-2. Synthesis of a tert-BMA Macromonomer

The t-BMA oligomer (5.00 g, 1.38 mmol) obtained in Step 8-1 wasdissolved in dimethylformamide (DMF) (50 ml). To the solution was addedaqueous 50% KOH (0.774 g) and, as a phase transfer catalyst,tetrabutylphosphonium bromide (TBPB) (0.468 g, 1.38 mmol). The resultantmixture was stirred for 24 hours at 30° C. Subsequently,chloromethylstyrene (4.24 g, 27.6 mmol) was added. The mixture wasallowed to react for 48 hours at 30° C. After reaction, the reactionsubstance was subjected to a purification step, in which reprecipitationwas performed through use of a 1:1 water-methanol mixture. Theintroduction rate of vinyl benzyl group was calculated based on the¹H-NMR data. As a result, it was confirmed that vinyl benzyl group hadbeen introduced almost quantitatively. The molecular weight (Mn) of theobtained macromonomer was determined to be 4,070 by GPC.

8-3. Copolymerization of the t-BMA Macromonomer and Styrene (Synthesisof a Graft Copolymer (B-i-1) and Preparation of Nanoparticles)

The t-BMA macromonomer obtained in Step 8-2 (0.300 g, 0.083 mmol) andstyrene (0.345 g, 3.32 mmol) were dissolved in ethanol (5 ml). AIBN, aradical polymerization initiator, was added (5.88 mg, 0.036 mmol)thereto, and copolymerization was performed in a deaerated sealed tubefor 48 hours at 60° C. After reaction, the contents of the tube werepurified by dialysis. Measurement by the dynamic light scatteringspectrophotometry revealed that the average particle size was 679 nm.

Reference Example 9 A Graft Copolymer having Polymethacrylic Acid as theGraft Chain (Graft Copolymer (B-1-2)):

The nanoparticles obtained in Step 8-3 were dispersed in 2N-HCl-ethanol,and hydrolyzed for 12 hours at 80° C., to thereby convert the ester ofthe macromonomer into carboxyl group. After reaction, the nanoparticleswere purified through dialysis. Measurement by the dynamic lightscattering spectrophotometry revealed that the average particle size ofthe nanoparticles was 835 nm.

Reference Example 10 A Graft Copolymer having Poly N-vinylacetamide asthe Graft Chain (Graft Copolymer (B-2-1))

10-1. Synthesis of an N-vinylacetamide Oligomer

An N-vinylacetamide (NVA) monomer (10 g, 117.6 mmol) was dissolved inethanol (50 ml). 2-Mercaptoethanol (2.3 g, 29.44 mmol) as a chaintransfer agent and azobisisobutyronitrile (0.197 g, 1.2 mmol) as aradical polymerization initiator were added thereto, and polymerizationwas performed for 6 hours at 60° C. in an atmosphere of nitrogen, tothereby synthesize an NVA oligomer. After reaction, the reactionsubstance was subjected to reprecipitation several times through use ofdiethylether for purification. The molecular weight (Mn) of the obtainedoligomer was determined to be 2,500 by GPC.

10-2. Synthesis of an NVA Macromonomer

The NVA oligomer (1.875 g, 0.75 mmol) obtained in Step 10-1 wasdissolved in dimethylformamide (50 ml). To the solution was addedaqueous 50% KOH (0.84 g, 7.5 mmol) and, as a phase transfer catalyst,tetrabutylphosphonium bromide (0.127 g, 0.374 mmol). The resultantmixture was stirred for 30 minutes, and subsequently,chloromethylstyrene (1.152 g, 7.5 mmol) was added. The mixture wasallowed to react for 48 hours at 30° C., to thereby obtain an NVAmacromonomer. After reaction, the reaction substance was subjected to apurification step, in which reprecipitation was performed several timesthrough use of diethylether. The introduction rate of vinyl benzyl groupwas calculated based on the ¹H-NMR data. As a result, it was confirmedthat vinyl benznyl group had been introduced almost quantitatively. Themolecular weight (Mn) of the obtained macromonomer was determined to be2,600 by GPC.

10-3. Copolymerization of the NVA Macromonomer and Styrene (Synthesis ofa Graft Copolymer (B-2-1); and Preparation of Nanoparticles)

The NVA macromonomer obtained in Step 10-2 (0.25 g, 0.096 mmol) andstyrene (0.23 ml, 1.99 mmol) were dissolved in ethanol (5 ml). A radicalpolymerization initiator azobisisobutyronitrile (3.34 mg, 0.02 mmol) wasadded, and copolymerization was performed in a deaerated sealed tube for48 hours at 60° C. After reaction, the contents of the tube weredialyzed so as to remove unreacted substances. Measurement by thedynamic light scattering spectrophotometry revealed that the averageparticle size of the graft copolymer was 257 nm.

Reference Example 11 Graft Copolymer having Polyvinylamine as the GraftChain (Graft Copolymer (B-2-2))

The nanoparticles obtained in Step 10-3 were dispersed in 2N-HCl, andhydrolyzed for 12 hours at 100° C., to thereby hydrolyze the amide bondin the macromonomer chain. The nanoparticles, after reaction, werepurified through dialysis. Measurement by the dynamic light scatteringspectrophotometry revealed that the average particle size of thenanoparticles was 273 nm.

Example 1 Preparation of a Complex of Nanoparticles andPhenolsulfonphthalein (PSP) (a Nanoparticle Preparation of PSP)

A monosodium salt of PSP (PSP-Na) was dissolved in phosphate buffer (pH7.0, 0.50 mM) containing sucrose at a concentration of 3.15 w/v %, sothat the concentration of the PSP-Na was 20 mg/ml. To the solution wasadded the freeze-dried product (graft copolymer (A-1)) obtained in Step1-3 of Reference Example 1 at a concentration of 20 mg/ml. The mixturewas brought to a uniform dispersion, to thereby obtain a nanoparticlepreparation. Independently, an aqueous PSP-Na solution in whichnanoparticles were not dispersed were prepared in a similar manner, andthis product was used as a control preparation.

Example 2 In vivo Evaluation of the Complex of Nanoparticles andPhenolsulfonphthalein (PSP)

2-1. Method

SD male rats (7 weeks old, about 200 g) were fasted for 24 hours. Underetherization, laparotomy was performed. By use of an injection needle,which was inserted through the pyrolus, the nanoparticle preparation(0.5 ml) prepared in Example 1 or the control preparation (0.5 ml) wasadministered duodenumally to the rats (dose: 9.4 mg in terms of PSP perrat; n=6). The cut was closed immediately after administration. Bloodwas collected from the carotid artery at 0.5, 1, 2, 4, 8, 12, and 24hours following the administration.

In accordance with the method of K. Higaki (Journal of PharmaceuticalScience, 79, 334, 1990), the plasma PSP concentration of each rat wasmeasured. The plasma (0.3 ml) that had been centrifugally separated fromthe collected blood, purified water (0.3 ml), and aqueous 0.1 N NaOHsolution (0.9 ml) were mixed. The mixture was filtered through use of anultrafilter membrane (molecular weight of fractionation=10,000), tothereby remove proteins, etc. The filtrate was used as a samplesolution. Separately, standard solutions were prepared through use ofaqueous PSP-Na solutions (starting from 9.4 mg/ml in terms of PSP, aseries of 2-fold dilutions) (0.3 ml each), a blank plasma from each rat(0.3 ml), and aqueous 0.1 N NaOH solution (0.9 ml) were mixed andultrafiltered. For each of the sample solutions and standard solutions,absorption was determined through use of a spectrometer at thewavelength of 560 nm. Based on the results obtained from the standardsolutions, a calibration curve was obtained, and the plasma PSPconcentrations were measured. Based on the obtained time-plasma PSPconcentration profile, pharmacokinetic parameters were calculated.

2-2. Results

The time-plasma PSP concentration profile is shown in FIG. 6, and thecalculated pharmacokinetic parameters are shown in Table 1. As isapparent from Table 1, the mean residence time (MRT) of PSP wassignificantly increased when PSP was mixed with nanoparticles (level ofsignificance: 1%). Thus, it was proved that the nanoparticle preparation(i.e, a complex of nanoparticles and PSP) making use of the graftcopolymer of the present invention has controlled release properties.

TABLE 1 (mean ± S.D.) AUC_(0-inf) C_(max) t_(max) MRT_(0-24 h) Aq.PSP-Na 9.97 ± 2.20 2.00 ± 0.29 0.92 ± 0.20 4.57 ± 0.34 sol. (controlprep.) Complex of 9.53 ± 1.15 1.28 ± 0.32 0.58 ± 0.20 6.74 ± 0.38 PSP &nano- particles (Example 1)

Example 3 Preparation of a Complex of Nanoparticles and SalmonCalcitonin (sCT) (a Nanoparticle Preparation of sCT)

An aqueous sCT solution having a concentration of 200 μg/ml and adispersion (dispersion medium: water) of the nanoparticles obtained inStep 1-3 of Reference Example 1 (graft copolymer (A-1)), which had aconcentration of 60 mg/ml, were independently prepared. The two weremixed in equal amounts and the nanoparticles were dispersed uniformly,to thereby obtain a nanoparticle preparation. Separately, an aqueous sCTsolution in which nanoparticles were not dispersed was prepared in amanner similar to that described above, to thereby obtain a controlpreparation.

Example 4 In vivo Evaluation of the Complex of Nanoparticles and SalmonCalcitonin (sCT)

4-1. Method

To SD male rats (7 weeks old, about 200 g) fasted for 24 hours wereadministered perorally the nanoparticle preparation (0.5 ml) prepared inExample 3 or the control preparation (0.5 ml) (n=5). Afteradministration, blood was collected from the tail vein of each rat in anamount of about 60 μl at 40 minutes, 80 minutes, 2, 3, 4, 6, and 8 hoursfollowing the administration.

Using a 634 Ca⁺⁺/pH analyzer (Ciba-Coning), the concentration of ionizedcalcium in the collected blood was measured. The difference between theionized calcium concentration at time 0 and that at each of theaforementioned times was calculated and plotted. From the plots,presence or absence of the effect of absorption enhancement obtainedthrough formation of nanoparticles was determined.

In this connection, sCT is known to have the pharmacological effect ofreducing the blood ionized calcium concentration.

4-2. Results

The results are shown in FIG. 7. As is apparent from FIG. 7, the bloodionized calcium concentration was slightly decreased when the aqueoussCT solution (i.e., a control preparation) was administered, howeverthis effect of reducing the blood ionized calcium concentration wasconsiderably enhanced when the nanoparticle preparation (a complex ofnanoparticles and sCT) obtained in Example 3 was administered. Theeffect enhanced by the invention nanoparticle preparation persisteduntil 8 hours had passed after administration.

Thus, it was confirmed that the nanoparticle preparation making use ofthe graft copolymer of the present invention improves gastrointestinalabsorption of sCT.

Example 5 Preparation of a Complex of Nanoparticles and Opioid Peptide(OP) (a Nanoparticle Preparation of OP)

An aqueous opioid peptide solution having a concentration of 200 μg/mland a dispersion (dispersion medium: water) of the nanoparticlesobtained in Step 1-3 of Reference Example 1 (graft copolymer (A-1)),which had a concentration of 20 mg/ml, were independently prepared. Thetwo were mixed in equal amounts and the nanoparticles were disperseduniformly, to thereby obtain a nanoparticle preparation (a complex ofnanoparticles and opioid peptide). Separately, an aqueous opioid peptidesolution in which nanoparticles were not dispersed was prepared in amanner similar to that described above, to thereby obtain a controlpreparation (100 μg/ml opioid peptide). The chemical structure of theopioid peptide used in this Example is as follows:

H₃CC(NH)-Tyr-D-Arg-Phe-N(CH₃)-β-Ala

Example 6 In vivo Evaluation of the Complex of Nanoparticles and OpioidPeptide (OP)

6-1. Method

To each of ddy male mice (3-4 weeks old, about 20-25 g) that were freelyfed was administered perorally the nanoparticle preparation prepared inExample 5 or the control preparation (opioid pepetide: 1 mg/10 ml/kg).Pressure stimulus was applied to the basal part of the mouse's tail byuse of a Randall & Selitto pressure-imparting apparatus (model MK-300,Muromachi Kikai), (32 g/second). Pain threshold values (g) were measuredat several points of time (1, 2, 3, 4, 5, 6, 8, and 24 hrs.) by use ofstruggling, biting of the stimulated part, and similar behaviors asindices. The cut-off value was 500 g. The pain-related activity (% ofMPE (maximum possible effect) was calculated according to the followingequation:

% of MPE={(Pain threshold value after administration)−(Pain thresholdvalue before administration)/(500−Pain threshold value beforeadministration)}×100

6-2. Results

The results are shown in FIG. 8. As is apparent from FIG. 8, thepain-related activity (% of MPE) was confirmed to be enhanced by mixingthe opioid peptide with nanoparticles.

Thus, it was confirmed that the nanoparticle preparation (a complex ofnanoparticles and opioid peptide) making use of the graft copolymer ofthe present invention improves gastrointestinal absorption of opioidpeptide.

Example 7 Preparation of a Complex of Nanoparticles and SalmonCalcitonin (sCT)

An aqueous sCT solution having a concentration of 200 μ/ml and adispersion (dispersion medium: water) of the nanoparticles obtained inReference Examples 2 and 4 (graft copolymers (A-2) and (A-4)), which hada concentration of 20 mg/ml, were independently prepared. The two weremixed in equal amounts and the nanoparticles were dispersed uniformly,to thereby obtain a nanoparticle preparation. Separately, an aqueous sCTsolution in which nanoparticles were not dispersed was prepared in amanner similar to that described above, to thereby obtain a controlpreparation.

Example 8 In vivo Evaluation of the Complex of Nanoparticles and SalmonCalcitonin (sCT)

By use of the nanoparticle preparation or the control preparationobtained in Example 7, the procedure of Example 4 was repeated tothereby determine the presence or absence of the effect of absorptionenhancement obtained by the formation of nanoparticles. The results areshown in FIG. 9.

As is apparent from FIG. 9, the blood ionized calcium concentration wasslightly decreased when the aqueous sCT solution (i.e., a controlpreparation) was administered, however this effect of reducing the bloodionized calcium concentration was considerably enhanced when thenanoparticle preparation (a complex of nanoparticles and sCT) obtainedin Example 7 was administered. The effect enhanced by the inventionnanoparticle preparation persisted until 6 hours had passed afteradministration.

Thus, it was confirmed that the nanoparticle preparation making use ofthe graft copolymer of the present invention improves gastrointestinalabsorption of sCT.

Example 9 Preparation of a Complex (which is a Nanoparticle Preparation)of a Mixture of Two Different Nanoparticles and Salmon Calcitonin (sCT)

An aqueous sCT solution having a concentration of 0.1 mg/ml, adispersion (dispersion medium: water) of the nanoparticles obtained inStep 1-3 of Reference Example 1 (graft copolymer (A-1)), which had aconcentration of 5 mg/ml, and a dispersion (dispersion medium: water) ofthe nanoparticles obtained in Reference Example 11 (graft copolymer(B-2-2)), which had a concentration of 5 mg/ml were independentlyprepared. The three were mixed in equal amounts and the nanoparticleswere dispersed uniformly, to thereby obtain a nanoparticle preparation.Separately, an aqueous sCT solution in which nanoparticles were notdispersed was prepared in a manner similar to that described above, tothereby obtain a control preparation.

Example 10 In vivo Evaluation of the Mixture of Nanoparticles and SalmonCalcitonin (sCT)

By use of the nanoparticle preparation or the control preparationobtained in Example 9, the procedure of Example 4 was repeated tothereby determine the presence or absence of the effect of absorptionenhancement obtained by the formation of nanoparticles. The results areshown in FIG. 10.

As is apparent from FIG. 10, the blood ionized calcium concentration wasslightly decreased when the aqueous sCT solution (i.e., a controlpreparation) was administered, however this effect of reducing the bloodionized calcium concentration was considerably enhanced when thenanoparticle preparation (a complex of two different kinds ofnanoparticles and sCT) obtained in Example 9 was administered. Theeffect enhanced by the invention nanoparticle preparation persisteduntil 5 hours had passed after administration. Moreover, the effect waseven further enhanced as compared to the case in which a single kind ofnanoparticles was used.

Thus, it was determined that the nanoparticle preparation making use oftwo or more kinds of the graft copolymer of the present inventionimproves gastrointestinal absorption of sCT.

Example 11 Divided Administration of a Nanoparticle Preparation

A test was performed to investigate the absorption enhancement effectattained by a nanoparticle preparation when administered twice individed amounts, with a time interval of 40 minutes.

An aqueous sCT solution having a concentration of 100 μg/ml and adispersion (dispersion medium: water) of the nanoparticles obtained inStep 1-3 of Reference Example 1 (graft copolymer (A-1)), whichdispersion had a concentration of 10 mg/ml, were independently prepared.The two were mixed in equal amounts and the nanoparticles were disperseduniformly, to thereby obtain a nanoparticle preparation. Separately, anaqueous sCT solution in which nanoparticles were not dispersed wasprepared in a manner similar to that described above, to thereby obtaina control preparation.

The procedure of Example 4 was repeated except that each preparation(0.5 ml) was divided into two (0.25 ml×2) and the divided portions wereadministered at time 0 and at 40 minutes, to thereby investigate theabsorption enhancement effect of salmon calcitonin.

As a result, as is apparent from FIG. 11, it was confirmed that thenanoparticle preparation of the present invention exerted an evenimproved effect of enhancing sCT absorption, when administered individed amounts with a certain time interval.

INDUSTRIAL APPLICABILITY

The pharmaceutical composition of the present invention that makes useof the particulate carriers exhibits an excellent peroral absorptionenhancement effect of the drug incorporated in the composition.Therefore, it is particularly useful as a DDS for poor absorptive drugs.

What is claimed is:
 1. A pharmaceutical composition, comprising: a drug;and a graft copolymer (A) consisting essentially of structural units ofthe following formulae (1) and (2):

wherein Q¹ is a hydrogen atom, a methyl group, or a cyano group, and Q²is a hydrogen atom,

wherein R¹ is a hydrogen atom or a halogenomethyl group, R² is a C₁-C₁₀alkyl group, R³ is a hydrogen atom or a C₁-C₁₀ alkyl group, and R⁴ is aC₁-C₁₀ alkyl group, provided that the total carbon number of R³ and R⁴taken together is between 3 and 20 inclusive;

wherein Q³ is a hydrogen atom or a methyl group, Q⁴ is a group havingthe following structure:

wherein A¹ is a C₁-C₁₀ alkylene group, Q⁵ is an oxygen atom, Q⁶ is aC₁-C₁₀ alkylene group, Q⁷ is an oxygen atom or a sulfur atom, X¹ is twohydrogen atoms, each of R⁵, R⁷, and R⁸ is a hydrogen atom or a methylgroup, R⁶ is a C₁-C₁₀ alkyl group, l is a number from 1 to 100, and eachof m and n is a number from 0 to 100; wherein the mole fraction of thestructural unit of formula (2) in graft copolymer (A) is between 0.001and
 1. 2. A pharmaceutical composition, comprising; a drug; and thefollowing components (a) and (b): (a) a graft copolymer (A) consistingessentially of structural units of the following formulae (1) and (2):

wherein Q¹ is a hydrogen atom, a methyl group, or a cyano group, and Q²is a hydrogen atom,

wherein R¹ is a hydrogen atom or a halogenomethyl group, R² is a C₁-C₁₀alkyl group, R³ is a hydrogen atom or a C₁-C₁₀ alkyl group, and R⁴ is aC₁-C₁₀ alkyl group, provided that the total carbon number of R³ and R⁴taken together is between 3 and 20 inclusive;

wherein Q³ is a hydrogen atom or a methyl group, Q⁴ is a group havingthe following structure:

wherein A¹ is a C₁-C₁₀ alkylene group, Q⁵ is an oxygen atom, Q⁶ is aC₁-C₁₀ alkylene group, Q⁷ is an oxygen atom or a sulfur atom, X¹ is twohydrogen atoms, each of R⁵, R⁷, and R⁸ is a hydrogen atom or a methylgroup, R⁶ is a C₁-C₁₀ alkyl group, l is a number from 1 to 100, and eachof m and n is a number from 0 to 100; and (b) one or more graftcopolymers selected from the group consisting of the following graftcopolymers (B-1) and (B-2): (B-1) a graft copolymer consistingessentially of structural units of the following formulae (1) and (3):

wherein Q¹ is a hydrogen atom, a methyl group, or a cyano group, and Q²is a hydrogen atom,

wherein R¹ is a hydrogen atom or a halogenomethyl group, R² is a C₁-C₁₀alkyl group, R³ is a hydrogen atom or a C₁-C₁₀ alkyl group, and R⁴ is aC₁-C₁₀ alkyl group, provided that the total carbon number of R³ and R⁴taken together is between 3 and 20 inclusive;

wherein Q⁸ is a hydrogen atom or a methyl group, Q⁹ is a group havingthe following structure:

wherein A² is a C₁-C₁₀ alkylene group, Q¹⁰ is an oxygen atom, Q¹¹ is aC₁-C₁₀ alkylene group, Q¹² is an oxygen atom or a sulfur atom, X² is twohydrogen atoms, each of R⁹ and R¹⁰ is a hydrogen atom or a methyl group,R¹¹ is a C₁-C₁₀ alkyl group, and p and q are independently numbers from0 to 100 such that the sum p+q is greater than or equal to 1; (B-2) agraft copolymer consisting essentially of structural units of thefollowing formulae (1) and (4):

wherein Q¹ is a hydrogen atom, a methyl group, or a cyano group, and Q²is a hydrogen atom,

wherein R¹ is a hydrogen atom or a halogenomethyl group, R² is a C₁-C₁₀alkyl group, R³ is a hydrogen atom or a C₁-C₁₀ alkyl group, and R⁴ is aC₁-C₁₀ alkyl group, provided that the total carbon number of R³ and R⁴taken together is between 3 and 20 inclusive;

wherein Q¹³ is a hydrogen atom or a methyl group, Q¹⁴ is a group havingthe following structure:

wherein A³ is a C₁-C₁₀ alkylene group, Q¹⁵ is an oxygen atom, Q¹⁶ is aC₁-C₁₀ alkylene group, Q¹⁷ is an oxygen atom or a sulfur atom, X³ is twohydrogen atoms, each of R¹² and R¹³ is a hydrogen atom or a methylgroup, R¹⁴ is a C₂-C₁₁ alkanoyl group, and s and t are independentlynumbers from 0 to 100 such that the sum s+t is greater than or equal to1; and wherein: the mole fraction of the structural unit of formula (2)in graft copolymer (A) is between 0.001 and 1, the mole fraction of thestructural unit of formula (3) in graft copolymer (B-1) is between 0.001and 1, and the mole fraction of the structural unit of formula (4) ingraft copolymer (B-2) is between 0.001 and
 1. 3. A pharmaceuticalcomposition, comprising: a complex of a graft copolymer A and a drug;wherein said graft copolymer (A) consists essentially of structuralunits of the following formulae (1) and (2):

wherein Q¹ is a hydrogen atom, a methyl group, or a cyano group, and Q²is a hydrogen atom,

wherein R¹ is a hydrogen atom or a halogenomethyl group, R² is a C₁-C₁₀alkyl group, R³ is a hydrogen atom or a C₁-C₁₀ alkyl group, and R⁴ is aC₁-C₁₀ alkyl group, provided that the total carbon number of R³ and R⁴taken together is between 3 and 20 inclusive;

wherein Q³ is a hydrogen atom or a methyl group, Q⁴ is a group havingthe following structure:

wherein A¹ is a C₁-C₁₀ alkylene group, Q⁵ is an oxygen atom, Q⁶ is aC₁-C₁₀ alkylene group, Q⁷ is an oxygen atom or a sulfur atom, X¹ is twohydrogen atoms, each of R⁵, R⁷, and R⁸ is a hydrogen atom or a methylgroup, R⁶ is a C₁-C₁₀ alkyl group, l is a number from 1 to 100, and eachof m and n is a number from 0 to 100; wherein the mole fraction of thestructural unit of formula (2) in graft copolymer (A) is between 0.001and
 1. 4. A pharmaceutical composition, comprising: a complex of a graftcopolymer composition; and a drug; wherein said graft copolymercomposition comprises the following components (a) and (b): (a) a graftcopolymer (A) consisting essentially of structural units of thefollowing formulae (1) and (2):

wherein Q¹ is a hydrogen atom, a methyl group, or a cyano group, and Q²is a hydrogen atom,

wherein R¹ is a hydrogen atom or a halogenomethyl group, R² is a C₁-C₁₀alkyl group, R³ is a hydrogen atom or a C₁-C₁₀ alkyl group, and R⁴ is aC₁-C₁₀ alkyl group, provided that the total carbon number of R³ and R⁴taken together is between 3 and 20 inclusive;

wherein Q³ is a hydrogen atom or a methyl group, Q⁴ is a group havingthe following structure:

wherein A¹ is a C₁-C₁₀ alkyl group, Q⁵ is an oxygen atom, Q⁶ is a C₁-C₁₀alkylene group, Q⁷ is an oxygen atom or a sulfur atom, X¹ is twohydrogen atoms, each of R⁵, R⁷, and R⁸ is a hydrogen atom or a methylgroup, R⁶ is a C₁-C₁₀ alkyl group, l is a number from 1 to 100, and eachof m and n is a number from 0 to 100; and (b) one or more graftcopolymers selected from the group consisting of the following graftcopolymers (B-1) and (B-2): (B-1) a graft copolymer consistingessentially of structural units of the following formulae (1) and (3):

wherein Q¹ is a hydrogen atom, a methyl group, or a cyano group, and Q²is a hydrogen atom,

wherein R¹ is a hydrogen atom or a halogenomethyl group, R² is a C₁-C₁₀alkyl group, R³ is a hydrogen atom or a C₁-C₁₀ alkyl group, and R⁴ isalkyl group, provided that the total carbon number of R³ and R⁴ takentogether is between 3 and 20 inclusive;

wherein Q⁸ is a hydrogen atom or a methyl group, Q⁹ is a group havingthe following structure:

wherein A² is a C₁-C₁₀ alkylene group, Q¹⁰ is an oxygen atom, Q¹¹ is aC₁-C₁₀ alkylene group, Q¹² is an oxygen atom or a sulfur atom, X² is twohydrogen atoms, each of R⁹ and R¹⁰ is a hydrogen atom or a methyl group,R¹¹ is a C₁-C₁₀ alkyl group, and p and q are independently numbers from0 to 100 such that the sum p+q is greater than or equal to 1; (B-2) agraft copolymer consisting essentially of structural units of thefollowing formulae (1) and (4):

wherein Q¹ is a hydrogen atom, a methyl group, or a cyano group, and Q²is a hydrogen atom,

wherein R¹ is a hydrogen atom or a halogenomethyl group, R² is a C₁-C₁₀alkyl group, R³ is a hydrogen atom or a C₁-C₁₀ alkyl group, and R⁴ is aC₁-C₁₀ alkyl group, provided that the total carbon number of R³ and R⁴taken together is between 3 and 20 inclusive;

wherein Q¹³ is a hydrogen atom or a methyl group, Q¹⁴ is a group havingthe following structure:

wherein A³ is a C₁-C₁₀ alkylene group, Q¹⁵ is an oxygen atom, Q¹⁶ is aC₁-C₁₀ alkyl group, Q¹⁷ is an oxygen atom or a sulfur atom, X³ is twohydrogen atoms, each of R¹² and R¹³ is a hydrogen atom or a methylgroup, R¹⁴ is a C₂-C₁₁ alkanoyl group, and s and t are independentlynumbers from 0 to 100 such that the sum s+t is greater than or equal to1; and wherein: the mole fraction of the structural unit of formula (2)in graft copolymer (A) is between 0.001 and 1, the mole fraction of thestructural unit of formula (3) in graft copolymer (B-1) is between 0.001and 1, and the mole fraction of the structural unit of formula (4) ingraft copolymer (B-2) is between 0.001 and
 1. 5. The pharmaceuticalcomposition according to claim 1, wherein the drug is a poor absorptivedrug.
 6. The pharmaceutical composition according to claim 1, whereinthe drug is a peptide drug.
 7. The pharmaceutical composition accordingto claim 6, wherein the peptide drug is an opioid peptide or calcitonin.8. The pharmaceutical composition according to claim 2, wherein saiddrug is a poor absorptive drug.
 9. The pharmaceutical compositionaccording to claim 2, wherein said drug is a peptide drug.
 10. Thepharmaceutical composition according to claim 9, wherein said peptidedrug is an opioid peptide or calcitonin.
 11. The pharmaceuticalcomposition according to claim 3, wherein said drug is a poor absorptivedrug.
 12. The pharmaceutical composition according to claim 3, whereinsaid drug is a peptide drug.
 13. The pharmaceutical compositionaccording to claim 12, wherein said peptide drug is an opioid peptide orcalcitonin.
 14. The pharmaceutical composition according to claim 4,wherein said drug is a poor absorptive drug.
 15. The pharmaceuticalcomposition according to claim 4, wherein said drug is a peptide drug.16. The pharmaceutical composition according to claim 15, wherein saidpeptide drug is an opioid peptide or calcitonin.
 17. The pharmaceuticalcomposition according to claim 1, wherein said drug is a poor absorptivedrug.
 18. The pharmaceutical composition according to claim 1, whereinsaid drug is a peptide drug.
 19. The pharmaceutical compositionaccording to claim 18, wherein said peptide drug is an opioid peptide orcalcitonin.